Anthrax toxin protective antigen protein (PA, 83 kDa) binds to receptors on the surface of mammalian cells, is cleaved by the cell surface protease furin, and then captures either of the two other toxin proteins, lethal factor (LF, 90 kDa) or edema factor (EF, 89 kDa). The PA-LF and PA-EF complexes enter cells by endocytosis and LF and EF translocate to the cytosol. EF is a calcium- and calmodulin-dependent adenylyl cyclase that causes large and unregulated increases in intracellular cAMP concentrations. LF is a metalloprotease that cleaves several mitogen-activated protein kinase kinases (MEKs). Entry of anthrax toxin into cells depends on two related cell surface receptors, tumor endothelium marker 8 (TEM8) and capillary morphogenesis gene product 2 (CMG2). TEM8 was initially identified as a protein upregulated in colon cancers. CMG2 has substantial sequence similarity to this candidate tumor marker. The tissue distribution and the relative importance of the two toxin receptors in toxin action are not well understood. During 2012, in collaboration with an Indian scientist having a long association with NIH and our laboratory, we identified two enzymes produced by Bacillus anthracis as being dual specificity protein kinases (DSPKs), unique enzymes that autophosphorylate on Ser, Thr, and Tyr residues and phosphorylate substrates on Ser and Thr residues. Transcriptional analyses of these kinases, Bas2152 (PrkD) and Bas2037 (PrkG), showed that they are expressed in all phases of growth. PrkD was found to be similar to the eukaryotic dual specificity Tyr phosphorylation- regulated kinase class of dual specificity kinases, which autophosphorylates on Ser, Thr, and Tyr residues and phosphorylates Ser and Thr residues on substrates. PrkG was found to be a bona fide dual specificity protein kinase that mediates autophosphorylation and substrate phosphorylation on Ser, Thr, and Tyr residues. The sites of phosphorylation in both of the kinases were identified through mass spectrometry. Phosphorylation on Tyr residues regulates the kinase activity of PrkD and PrkG. PrpC, the only known B. anthracis Ser/Thr protein phosphatase, was also found to possess dual specificity. Genistein, a known Tyr kinase inhibitor, was found to inhibit the activities of PrkD and PrkG and affect the growth of B. anthracis cells, indicating a possible role of these kinases in cell growth and development. In addition, the glycolytic enzyme pyruvate kinase was found to be phosphorylated by PrkD on Ser and Thr residues but not by PrkG. Thus, this study provides the first evidence of DSPKs in B. anthracis that belong to different classes and have different modes of regulation. This reporting period also saw an important advance in our long-standing efforts to characterize the structure, function, and role in pathogenesis of the edema factor (EF) protein of anthrax toxin. In the current effort, anti-EF monoclonal antibodies (MAb) were produced following immunization of mice, and four of the antibodies were fully characterized. MAb 3F2 has an affinity of 388 pM, was most effective for EF detection, and appears to be the first antibody reported to neutralize EF by binding to the catalytic domain. MAb 7F10 shows potent neutralization of edema toxin activity in vitro and in vivo; it targets the N-terminal protective antigen-binding domain. The four MAb react with three different domains of edema factor, and all were able to detect purified edema factor by western blot analysis. None of the four MAb cross-reacted with the lethal factor toxin component. Three of the four MAb protected mice in both a systemic edema toxin challenge model and a subcutaneous spore challenge edema model. A combination of three of the MAb significantly delayed the time to death in a mouse infection model involving spore injection in the neck. The protection observed in this work appears to be the first direct evidence that monoclonal antibody-mediated neutralization of EF alone is sufficient to delay anthrax disease progression. In the year of 2012, we have also contributed in studies developing novel monoclonal antibodies to anthrax toxin protective antigen. These antibodies aid in structure-function analysis of the toxin proteins and can for the basis of therapeutics. We reported a practical strategy for development of simple antitoxins having substantial advantages over currently-available treatments. The strategy employs a single recombinant 'targeting agent' that binds a toxin at two unique sites and a 'clearing Ab' that binds two epitopes present on each targeting agent. Co-administration of the targeting agent and the clearing Ab results in decoration of the toxin with up to four Abs to promote accelerated clearance. The therapeutic strategy was applied to two botulinum neurotoxin (BoNT) serotypes and protected mice from lethality in two different intoxication models with an efficacy equivalent to conventional antitoxin serum. Targeting agents were a single recombinant protein consisting of a heterodimer of two camelid anti-BoNT heavy-chain-only Ab V(H) (VHH) binding domains and two E-tag epitopes. The clearing mAb was an anti-E-tag mAb. By comparing the in vivo efficacies of treatments that employed neutralizing vs. non-neutralizing agents or the presence vs. absence of the clearing Ab permitted unprecedented insight into the roles of toxin neutralization and clearance in antitoxin efficacy. Surprisingly, when a post-intoxication treatment model was used, a toxin-neutralizing heterodimer agent fully protected mice from intoxication even in the absence of clearing Ab. Thus a single, easy-to-produce recombinant protein was as efficacious as polyclonal antisera in a clinically-relevant mouse model of botulism. This strategy should have widespread application in antitoxin development and other therapies in which neutralization and/or accelerated clearance of a serum biomolecule can offer therapeutic benefit. A collaborative project completed during 2012 used knowledge and reagents developed in earlier studies on the role of cellular proteases in activation of bacterial protein toxins. These tools were used to identify cellular determinants controlling the infection of cells by adeno-associated viruses (AAV), which are the basis of viral vectors preferred in certain gene therapy applications. To identify these critical cellular determinants, we took advantage of the gene transfer abilities of AAV in combination with a forward genetic selection to identify proteins critical for transduction by this virus. AAV serotype 5 (AAV5) vectors encoding the furin gene were used to transduce furin-deficient CHO FD11 cells, followed by selection with furin-dependent bacterial protein toxins. A small number of spontaneously mutated cells specifically resistant to AAV5 transduction (and therefore surviving the toxin treatment) was isolated. Sequence analysis showed that they all had a single amino acid mutation in the leader sequence of the platelet-derived growth factor receptor alpha (PDGFRalpha) gene, which was previously shown to the be AAV receptor. Characterization of this mutation showed that it inhibited PDGFRalpha trafficking, resulting in limited expression on the plasma membrane. Mutagenesis and transfection experiments confirmed the effect of this mutation on PDGFRalpha trafficking, and the AAV5 resistant phenotype could be rescued by transfection with wild type PDGFRalpha. Thus, the tools of furin-deficient cells and selection with bacterial toxins enabled a genetic screen that identified interesting cellular genes controlling viral sensitivity.